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Studies on haemoglobin from the Indian toad, Bufo melanostictus , by electron microscopy
Author(s) -
Misra D. N.
Publication year - 1971
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1971.tb02282.x
Subject(s) - chemistry , uranyl acetate , crystallography , negative stain , molecule , electron microscope , hemoglobin , analytical chemistry (journal) , chromatography , ultrastructure , biology , anatomy , biochemistry , optics , physics , organic chemistry
SUMMARY Hæmoglobin from an amphibian, Bufo melanostictus , was studied by electron microscopy after metal shadowing and after negative staining with uranyl acetate. The effect of the electron stain on the protein was investigated by spectrophotometry. The sample was also characterized by alkali denaturation and sedimentation analysis. The results were compared to those of adult human hæmoglobin. Spectrophotometry of the sample showed characteristics similar to those of normal oxyhæmoglobin. The protein, like human adult hæmoglobin, was found to be completely alkali‐labile. Ultracentrifugation of a freshly prepared sample showed a single symmetrical peak, an indication for the presence of a homogeneous component. The S 0 20,W value (5.1) was found to be markedly higher than that of adult human hæmoglobin. With storage, however, the peak became asymmetrical suggesting the formation of a limited amount of a heavier component. The electron micrographs of the shadowed preparations revealed a characteristic shape and ultrastructure of the protein molecules and their average height was found to be 5.5 nm. The micrographs of the negatively stained samples showed many particles with a substructure similar to that of the shadowed molecules and a mean dimension of 6.1 nm, a number of dimers of average length 6.0 nm and overall width 3.6 nm, and many globules of average diameter 3.6 nm. These were interpreted as whole, half and quarter hæmoglobin molecules, respectively. Both techniques revealed at the most four subunits in a molecule. The structure of the subunits and their arrangement in the molecule were found to be essentially similar to those of normal human adult hæmoglobin, and could be interpreted in terms of the Perutz model. Spectrophotometry of the mixture of the protein and uranyl acetate showed that the protein becomes denatured with time and that the denaturation is due to the low pH (4.3) of the stain. Nevertheless, this study showed that by keeping the staining time very short, it is possible to obtain acceptable electron micrographs of small multi‐unit protein molecules such as haemoglobin.